Metagenomic analysis of Limnohabitans population in Lake Michigan
Ruben Props
Today
# Read data
mean_coverage <- read.table("./anvio_output/mean_coverage_selected_final.tsv", header = TRUE)
std_coverage <- read.table("./anvio_output/std_coverage_selected_final.tsv", header = TRUE)
bin_size <- read.table("./anvio_output/general_bins_summary_selected_final.tsv", header = TRUE)[, c(2,4)]
total_reads <- read.table("./anvio_output/sample_reads.tsv", header = TRUE)
read_length <- 150
# From wide to long format
mean_coverage_long <- gather(mean_coverage, Sample_ID, coverage,
Fa13_BD_MLB_DN:Su13_BD_MM15_SN_C, factor_key=TRUE)
mean_coverage_long[,2] <- gsub(mean_coverage_long[,2], pattern = "_C",
replacement = "")
std_coverage_long <- gather(std_coverage, Sample_ID, std_coverage,
Fa13_BD_MLB_DN:Su13_BD_MM15_SN_C,
factor_key=TRUE)
std_coverage_long[,2] <- gsub(std_coverage_long[,2], pattern = "_C",
replacement = "")
coverage_data <- data.frame(mean_coverage_long,
std_coverage = std_coverage_long[,3])
# Read and add metadata
meta <- read.csv2("metadata.csv")
meta$Sample_ID <- gsub(meta$Sample_ID, pattern = ".", replacement = "_", fixed = TRUE)
data_total <- left_join(coverage_data, total_reads, by = c("Sample_ID" = "sample"))
data_total <- left_join(data_total, bin_size, by = "bins")
data_total <- left_join(data_total, meta, by = "Sample_ID")
# Calculate relative abundance of the bins
data_total$mean_rel_abundance <- 100*(data_total$coverage*data_total$bin_size)/(read_length*data_total$Total_reads)
data_total$upper_rel_abundance <- 100*((data_total$coverage+data_total$std_coverage)*data_total$bin_size)/(read_length*data_total$Total_reads)
data_total$lower_rel_abundance <- 100*((data_total$coverage-data_total$std_coverage)*data_total$bin_size)/(read_length*data_total$Total_reads)1a. Phylogenetic tree
RAxML tree for Limnohabitans genomes
1b. Network analysis based on 16S data
# import data
df_phy <- import_mothur(mothur_shared_file = "./16S/stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.an.unique_list.shared",
mothur_constaxonomy_file = "./16S/stability.trim.contigs.good.unique.good.filter.unique.precluster.pick.pick.an.unique_list.0.03.cons.taxonomy")
# Filter out 2013 samples
df_phy <- prune_samples(grep(pattern = ".", sample_names(df_phy), fixed = TRUE,
value = TRUE), df_phy)
df_phy <- prune_samples(grep(pattern = "cD", sample_names(df_phy), fixed = TRUE,
value = TRUE, invert = TRUE), df_phy)
# Perform prevalence filtering
df_phy <- filter_taxa(df_phy, function(x) sum(x > 30) > (0.25*length(x)), TRUE)
# Run spiec-easi
sp_easi <- spiec.easi(df_phy, method='mb', lambda.min.ratio=1e-2,
nlambda=20, icov.select.params=list(rep.num=50))## Normalizing/clr transformation of data with pseudocount ...## Inverse Covariance Estimation with mb ...## Model selection with stars ...## Done!ig.mb <- adj2igraph(sp_easi$refit, vertex.attr = list(name=taxa_names(df_phy)))
vsize <- Biobase::rowMax(clr(t(otu_table(df_phy)), 1))+10
Lineage_rel <- tax_table(df_phy)[,"Rank2"]
Lineage_rel <- factor(Lineage_rel, levels = unique(Lineage_rel))
# OTUs that are Limnohabitans
limno_otus <- taxa_names(df_phy)[tax_table(df_phy)[,"Rank6"] == "betI_A"]
limno_otus <- limno_otus[!is.na(limno_otus)]
# Make Limno label
limno_labs <- c()
limno_labs[vertex.attributes(ig.mb)$name %in% limno_otus] <- "Limnohabitans sp."
limno_labs[is.na(limno_labs)] <- ""
# Plot network
p_16S_network <- plot_network_custom(ig.mb, df_phy, type='taxa',
line_weight = 2, hjust = 0.5,
point_size = 0.1, alpha = 0.01, label=NULL, label_size = 3.95)+
scale_fill_brewer(palette = "Paired")+
scale_color_brewer(palette = "Paired")+
geom_point(aes(size = vsize, fill = Lineage_rel), alpha = 0.5,
colour="black", shape=21)+
guides(size = FALSE,
fill = guide_legend(title = "Phylum", override.aes = list(size = 5),
nrow = 4),
color = FALSE)+
theme(legend.position="bottom", legend.text=element_text(size=12),
text = element_text(size = 12),
plot.margin = unit(c(1,1,1,1), "cm"))+
scale_size(range = c(5, 15))+
geom_label_repel(aes(label = limno_labs), fontface = 'bold', color = 'black',
box.padding = 0.35, point.padding = 0.5,
segment.color = 'black',
size = 4,
# Width of the line segments.
segment.size = 1.5,
# Draw an arrow from the label to the data point.
arrow = arrow(length = unit(0.015, 'npc')),
nudge_x = -0.1,
nudge_y = 0.6
)
print(p_16S_network)
2. Investigate MAG- and 16S-based abundances
Formula used to calculate relative abundances: \[Relative\ abundance =100*(\frac{mean\ coverage * bin\ size}{read\ length*total\ sample\ reads })\]
# Plot abundance distributions of all bins
p_season <- ggplot(data = data_total, aes(x = bins, y = mean_rel_abundance, fill = bins))+
geom_point(size = 4, shape = 21, alpha = 0.7)+
geom_boxplot(alpha = 0.3)+
scale_fill_brewer(palette = "Paired")+
facet_grid(.~Site)+
theme_bw()+
geom_errorbar(aes(ymin=lower_rel_abundance,
ymax=upper_rel_abundance,
width=.1))+
facet_grid(Season~.)+
# ylim(0,1)+
theme(axis.text=element_text(size=14), axis.title=element_text(size=20),
title=element_text(size=20), legend.text=element_text(size=14),
legend.background = element_rect(fill="transparent"),
axis.text.x = element_text(angle = 45, hjust = 1),
strip.text.y=element_text(size=18))+
ylab("Mean relative abundance (%)")
p_station <- ggplot(data = data_total, aes(x = bins, y = mean_rel_abundance, fill = bins))+
geom_point(size = 4, shape = 21, alpha = 0.7)+
geom_boxplot(alpha = 0.3)+
scale_fill_brewer(palette = "Paired")+
facet_grid(.~Site)+
theme_bw()+
geom_errorbar(aes(ymin=lower_rel_abundance,
ymax=upper_rel_abundance,
width=.1))+
facet_grid(Site~.)+
# ylim(0,1)+
theme(axis.text=element_text(size=14), axis.title=element_text(size=20),
title=element_text(size=20), legend.text=element_text(size=14),
legend.background = element_rect(fill="transparent"),
axis.text.x = element_text(angle = 45, hjust = 1),
strip.text.y=element_text(size=18))+
ylab("Mean relative abundance (%)")
grid.arrange(p_season, p_station, nrow = 2)
3. Investigate sequence characteristics within coding DNA sequences (CDS)
# First we need the files that map the gene ID to the sequence ID (linux cmd: https://github.com/rprops/MetaG_lakeMI/wiki/11.-Genome-annotation)
# These are stored in the IMG_annotation data for each genome bin
# Next, extract the %GC of each gene from the gff file
Bin_2737471681 <- extract_gc_from_gff("./IMG_annotation/IMG_2737471681/IMG_Data/133052.assembled.gff",
outputFolder = "GC_analysis")
Bin_2737471682 <- extract_gc_from_gff("./IMG_annotation/IMG_2737471682/IMG_Data/133053.assembled.gff",
outputFolder = "GC_analysis")
Bin_2737471683 <- extract_gc_from_gff("./IMG_annotation/IMG_2737471683/IMG_Data/133054.assembled.gff",
outputFolder = "GC_analysis")
Bin_2737471793 <- extract_gc_from_gff("./IMG_annotation/IMG_2737471793/IMG_Data/133647.assembled.gff",
outputFolder = "GC_analysis")
Bin_2737471794 <- extract_gc_from_gff("./IMG_annotation/IMG_2737471794/IMG_Data/133648.assembled.gff",
outputFolder = "GC_analysis")
Bin_2737471795 <- extract_gc_from_gff("./IMG_annotation/IMG_2737471795/IMG_Data/133649.assembled.gff",
outputFolder = "GC_analysis")
Bin_2737471797 <- extract_gc_from_gff("./IMG_annotation/IMG_2737471797/IMG_Data/133651.assembled.gff",
outputFolder = "GC_analysis")
Bin_2737471799 <- extract_gc_from_gff("./IMG_annotation/IMG_2737471799/IMG_Data/133653.assembled.gff",
outputFolder = "GC_analysis")
Bin_2737471802 <- extract_gc_from_gff("./IMG_annotation/IMG_2737471802/IMG_Data/133656.assembled.gff",
outputFolder = "GC_analysis")
Bin_2737471804 <- extract_gc_from_gff("./IMG_annotation/IMG_2737471804/IMG_Data/133658.assembled.gff",
outputFolder = "GC_analysis")
Bin_2737471805 <- extract_gc_from_gff("./IMG_annotation/IMG_2737471805/IMG_Data/133659.assembled.gff",
outputFolder = "GC_analysis")
Bin_2737471806 <- extract_gc_from_gff("./IMG_annotation/IMG_2737471806/IMG_Data/133660.assembled.gff",
outputFolder = "GC_analysis")
# Use these files to make dataframes mapping function (COGs) and %GC
Bin_2737471681_gc_cog <- gc2function(seq_id_gc = "GC_analysis/seqid_GC_133052.assembled.gff.tsv", gene_id_seq_id ="./IMG_annotation/IMG_2737471681/IMG_Data/2737471681/2737471681.gene_oid_2_seq_id.txt",
functions = "./IMG_annotation/IMG_2737471681/IMG_Data/2737471681/2737471681.cog.tab.txt",
gc_thresh = 0.1, output = FALSE, label = "Bin_2737471681")## Wed Oct 18 15:25:24 2017 --- There are 2248 genes with > 0.1 %
## Wed Oct 18 15:25:24 2017 --- This is 100 % of all genes
## Wed Oct 18 15:25:24 2017 --- The 10 genes with the highest GC% are:
## function_id
## 2239 NADPH-dependent 2,4-dienoyl-CoA reductase, sulfur reductase, or a related oxidoreductase
## 2240 tRNA A37 threonylcarbamoyladenosine synthetase subunit TsaC/SUA5/YrdC
## 2241 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase
## 2242 Mg-chelatase subunit ChlD
## 2243 Enoyl-CoA hydratase/carnithine racemase
## 2244 Sugar or nucleoside kinase, ribokinase family
## 2245 DNA-binding transcriptional regulator, MarR family
## 2246 NAD(P)H-hydrate repair enzyme Nnr, NAD(P)H-hydrate dehydratase domain
## 2247 Peptidase E
## 2248 Sulfite exporter TauE/SafE
## function_name GC
## 2239 415 67.5
## 2240 211 67.6
## 2241 230 67.6
## 2242 261 68.0
## 2243 257 68.2
## 2244 311 68.2
## 2245 126 68.3
## 2246 284 68.9
## 2247 224 69.3
## 2248 232 69.4Bin_2737471682_gc_cog <- gc2function(seq_id_gc = "GC_analysis/seqid_GC_133053.assembled.gff.tsv", gene_id_seq_id ="./IMG_annotation/IMG_2737471682/IMG_Data/2737471682/2737471682.gene_oid_2_seq_id.txt",
functions = "./IMG_annotation/IMG_2737471682/IMG_Data/2737471682/2737471682.cog.tab.txt",
gc_thresh = 0.1, output = FALSE, label = "Bin_2737471682")## Wed Oct 18 15:25:24 2017 --- There are 1882 genes with > 0.1 %
## Wed Oct 18 15:25:24 2017 --- This is 100 % of all genes
## Wed Oct 18 15:25:24 2017 --- The 10 genes with the highest GC% are:
## function_id
## 1873 D-alanyl-D-alanine carboxypeptidase
## 1874 Aspartate/methionine/tyrosine aminotransferase
## 1875 DNA-binding transcriptional regulator, LysR family
## 1876 Predicted esterase of the alpha/beta hydrolase fold
## 1877 Sulfite exporter TauE/SafE
## 1878 Endonuclease YncB, thermonuclease family
## 1879 Predicted house-cleaning NTP pyrophosphatase, Maf/HAM1 superfamily
## 1880 L-aminopeptidase/D-esterase
## 1881 tRNA A37 threonylcarbamoyladenosine synthetase subunit TsaC/SUA5/YrdC
## 1882 Glycerate-2-kinase
## function_name GC
## 1873 470 68.0
## 1874 393 68.1
## 1875 297 68.1
## 1876 181 68.2
## 1877 232 68.3
## 1878 192 68.5
## 1879 193 68.9
## 1880 348 69.0
## 1881 211 69.5
## 1882 422 73.0Bin_2737471683_gc_cog <- gc2function(seq_id_gc = "GC_analysis/seqid_GC_133054.assembled.gff.tsv", gene_id_seq_id ="./IMG_annotation/IMG_2737471683/IMG_Data/2737471683/2737471683.gene_oid_2_seq_id.txt",
functions = "./IMG_annotation/IMG_2737471683/IMG_Data/2737471683/2737471683.cog.tab.txt",
gc_thresh = 0.1, output = FALSE, label = "Bin_2737471683")## Wed Oct 18 15:25:24 2017 --- There are 1727 genes with > 0.1 %
## Wed Oct 18 15:25:24 2017 --- This is 100 % of all genes
## Wed Oct 18 15:25:24 2017 --- The 10 genes with the highest GC% are:
## function_id
## 1718 Type II secretory pathway, component PulF
## 1719 Pyrroline-5-carboxylate reductase
## 1720 Shikimate kinase
## 1721 Phosphoribosylaminoimidazole carboxylase (NCAIR synthetase)
## 1722 ADP-heptose:LPS heptosyltransferase
## 1723 Cobalamin biosynthesis protein CobD/CbiB
## 1724 Acetolactate synthase large subunit or other thiamine pyrophosphate-requiring enzyme
## 1725 Uracil-DNA glycosylase
## 1726 NAD(P)-dependent dehydrogenase, short-chain alcohol dehydrogenase family
## 1727 tRNA A37 threonylcarbamoyladenosine synthetase subunit TsaC/SUA5/YrdC
## function_name GC
## 1718 397 63.1
## 1719 266 63.1
## 1720 172 63.2
## 1721 375 63.7
## 1722 334 63.8
## 1723 320 63.9
## 1724 550 64.0
## 1725 202 64.2
## 1726 251 64.4
## 1727 211 64.6Bin_2737471793_gc_cog <- gc2function(seq_id_gc = "GC_analysis/seqid_GC_133647.assembled.gff.tsv", gene_id_seq_id ="./IMG_annotation/IMG_2737471793/IMG_Data/2737471793/2737471793.gene_oid_2_seq_id.txt",
functions = "./IMG_annotation/IMG_2737471793/IMG_Data/2737471793/2737471793.cog.tab.txt",
gc_thresh = 0.1, output = FALSE, label = "Bin_2737471793")## Wed Oct 18 15:25:24 2017 --- There are 1476 genes with > 0.1 %
## Wed Oct 18 15:25:24 2017 --- This is 100 % of all genes
## Wed Oct 18 15:25:24 2017 --- The 10 genes with the highest GC% are:
## function_id
## 1467 Acetolactate synthase large subunit or other thiamine pyrophosphate-requiring enzyme
## 1468 2-keto-3-deoxy-L-rhamnonate aldolase RhmA
## 1469 ABC-type dipeptide/oligopeptide/nickel transport system, ATPase component
## 1470 Predicted Rossmann fold nucleotide-binding protein DprA/Smf involved in DNA uptake
## 1471 NAD(P)H-dependent flavin oxidoreductase YrpB, nitropropane dioxygenase family
## 1472 Pimeloyl-ACP methyl ester carboxylesterase
## 1473 CO or xanthine dehydrogenase, FAD-binding subunit
## 1474 tRNA A37 threonylcarbamoyladenosine modification protein TsaB
## 1475 Mg-chelatase subunit ChlD
## 1476 Uracil-DNA glycosylase
## function_name GC
## 1467 550 67.3
## 1468 255 67.4
## 1469 316 67.5
## 1470 350 67.7
## 1471 336 68.0
## 1472 282 68.0
## 1473 284 68.1
## 1474 220 68.3
## 1475 261 68.3
## 1476 202 69.2Bin_2737471794_gc_cog <- gc2function(seq_id_gc = "GC_analysis/seqid_GC_133648.assembled.gff.tsv", gene_id_seq_id ="./IMG_annotation/IMG_2737471794/IMG_Data/2737471794/2737471794.gene_oid_2_seq_id.txt",
functions = "./IMG_annotation/IMG_2737471794/IMG_Data/2737471794/2737471794.cog.tab.txt",
gc_thresh = 0.1, output = FALSE, label = "Bin_2737471794")## Wed Oct 18 15:25:25 2017 --- There are 1226 genes with > 0.1 %
## Wed Oct 18 15:25:25 2017 --- This is 100 % of all genes
## Wed Oct 18 15:25:25 2017 --- The 10 genes with the highest GC% are:
## function_id
## 1217 Cytochrome c-type biogenesis protein CcmH/NrfF
## 1218 Type II secretory pathway, component PulK
## 1219 Predicted ATPase with chaperone activity
## 1220 Geranylgeranyl pyrophosphate synthase
## 1221 Threonine/homoserine efflux transporter RhtA
## 1222 Predicted secreted protein
## 1223 Heme exporter protein D
## 1224 UDP-N-acetylmuramyl pentapeptide synthase
## 1225 Predicted Rossmann fold nucleotide-binding protein DprA/Smf involved in DNA uptake
## 1226 CO or xanthine dehydrogenase, FAD-binding subunit
## function_name GC
## 1217 153 66.0
## 1218 323 66.0
## 1219 490 66.1
## 1220 322 66.2
## 1221 292 66.3
## 1222 148 66.4
## 1223 67 66.7
## 1224 451 66.9
## 1225 350 67.9
## 1226 284 68.0Bin_2737471795_gc_cog <- gc2function(seq_id_gc = "GC_analysis/seqid_GC_133649.assembled.gff.tsv", gene_id_seq_id ="./IMG_annotation/IMG_2737471795/IMG_Data/2737471795/2737471795.gene_oid_2_seq_id.txt",
functions = "./IMG_annotation/IMG_2737471795/IMG_Data/2737471795/2737471795.cog.tab.txt",
gc_thresh = 0.1, output = FALSE, label = "Bin_2737471795")## Wed Oct 18 15:25:25 2017 --- There are 1383 genes with > 0.1 %
## Wed Oct 18 15:25:25 2017 --- This is 100 % of all genes
## Wed Oct 18 15:25:25 2017 --- The 10 genes with the highest GC% are:
## function_id
## 1374 Glycerol-3-phosphate dehydrogenase
## 1375 Predicted amidophosphoribosyltransferases
## 1376 2-polyprenyl-6-methoxyphenol hydroxylase and related FAD-dependent oxidoreductases
## 1377 DNA-binding transcriptional regulator, LysR family
## 1378 Tat protein secretion system quality control protein TatD (DNase activity)
## 1379 Phosphatidylglycerophosphate synthase
## 1380 Methylase of polypeptide chain release factors
## 1381 Methionyl-tRNA formyltransferase
## 1382 Tripartite-type tricarboxylate transporter, receptor component TctC
## 1383 Cytochrome c553
## function_name GC
## 1374 532 66.9
## 1375 225 67.0
## 1376 387 67.0
## 1377 297 67.1
## 1378 256 67.1
## 1379 192 67.5
## 1380 280 67.9
## 1381 307 68.0
## 1382 319 68.9
## 1383 121 69.4Bin_2737471797_gc_cog <- gc2function(seq_id_gc = "GC_analysis/seqid_GC_133651.assembled.gff.tsv", gene_id_seq_id ="./IMG_annotation/IMG_2737471797/IMG_Data/2737471797/2737471797.gene_oid_2_seq_id.txt",
functions = "./IMG_annotation/IMG_2737471797/IMG_Data/2737471797/2737471797.cog.tab.txt",
gc_thresh = 0.1, output = FALSE, label = "Bin_2737471797")## Wed Oct 18 15:25:25 2017 --- There are 1076 genes with > 0.1 %
## Wed Oct 18 15:25:25 2017 --- This is 100 % of all genes
## Wed Oct 18 15:25:25 2017 --- The 10 genes with the highest GC% are:
## function_id
## 1067 DNA-directed RNA polymerase specialized sigma subunit, sigma24 family
## 1068 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase
## 1069 tRNA A37 N6-isopentenylltransferase MiaA
## 1070 FAD/FMN-containing dehydrogenase
## 1071 Phosphoribosyl-ATP pyrophosphohydrolase
## 1072 ADP-heptose:LPS heptosyltransferase
## 1073 UDP-N-acetylmuramoylalanine-D-glutamate ligase
## 1074 Uncharacterized conserved protein YcfJ, contains glycine zipper 2TM domain
## 1075 2-polyprenyl-6-methoxyphenol hydroxylase and related FAD-dependent oxidoreductases
## 1076 Copper oxidase (laccase) domain
## function_name GC
## 1067 182 58.9
## 1068 160 58.9
## 1069 308 58.9
## 1070 459 59.1
## 1071 92 59.1
## 1072 334 59.5
## 1073 448 59.7
## 1074 179 59.7
## 1075 387 59.7
## 1076 249 60.0Bin_2737471799_gc_cog <- gc2function(seq_id_gc = "GC_analysis/seqid_GC_133653.assembled.gff.tsv", gene_id_seq_id ="./IMG_annotation/IMG_2737471799/IMG_Data/2737471799/2737471799.gene_oid_2_seq_id.txt",
functions = "./IMG_annotation/IMG_2737471799/IMG_Data/2737471799/2737471799.cog.tab.txt",
gc_thresh = 0.1, output = FALSE, label = "Bin_2737471799")## Wed Oct 18 15:25:25 2017 --- There are 522 genes with > 0.1 %
## Wed Oct 18 15:25:25 2017 --- This is 100 % of all genes
## Wed Oct 18 15:25:25 2017 --- The 10 genes with the highest GC% are:
## function_id
## 513 Glutathione S-transferase
## 514 DNA-nicking endonuclease, Smr domain
## 515 Carbonic anhydrase or acetyltransferase, isoleucine patch superfamily
## 516 Pyrroline-5-carboxylate reductase
## 517 16S rRNA U1498 N3-methylase RsmE
## 518 Thiamine monophosphate kinase
## 519 Nicotinamide mononucleotide (NMN) deamidase PncC
## 520 Pyrimidine reductase, riboflavin biosynthesis
## 521 Glucokinase
## 522 Nucleoside-diphosphate-sugar epimerase
## function_name GC
## 513 211 67.0
## 514 184 67.1
## 515 176 67.2
## 516 266 67.3
## 517 246 67.4
## 518 317 67.7
## 519 162 67.7
## 520 218 67.8
## 521 320 68.0
## 522 314 69.7Bin_2737471802_gc_cog <- gc2function(seq_id_gc = "GC_analysis/seqid_GC_133656.assembled.gff.tsv", gene_id_seq_id ="./IMG_annotation/IMG_2737471802/IMG_Data/2737471802/2737471802.gene_oid_2_seq_id.txt",
functions = "./IMG_annotation/IMG_2737471802/IMG_Data/2737471802/2737471802.cog.tab.txt",
gc_thresh = 0.1, output = FALSE, label = "Bin_2737471802")## Wed Oct 18 15:25:25 2017 --- There are 1691 genes with > 0.1 %
## Wed Oct 18 15:25:25 2017 --- This is 100 % of all genes
## Wed Oct 18 15:25:25 2017 --- The 10 genes with the highest GC% are:
## function_id
## 1682 Pyrimidine reductase, riboflavin biosynthesis
## 1683 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase
## 1684 CO or xanthine dehydrogenase, FAD-binding subunit
## 1685 Pyrimidine deaminase domain of riboflavin biosynthesis protein RibD
## 1686 Phosphohistidine phosphatase SixA
## 1687 Cyanate permease
## 1688 Mg-chelatase subunit ChlD
## 1689 Chromate transport protein ChrA
## 1690 Uncharacterized conserved protein YcbX, contains MOSC and Fe-S domains
## 1691 Predicted ATPase with chaperone activity
## function_name GC
## 1682 218 67.8
## 1683 230 68.0
## 1684 284 68.2
## 1685 146 68.3
## 1686 163 68.3
## 1687 395 69.1
## 1688 261 69.2
## 1689 195 69.4
## 1690 270 69.6
## 1691 490 70.7Bin_2737471804_gc_cog <- gc2function(seq_id_gc = "GC_analysis/seqid_GC_133658.assembled.gff.tsv", gene_id_seq_id ="./IMG_annotation/IMG_2737471804/IMG_Data/2737471804/2737471804.gene_oid_2_seq_id.txt",
functions = "./IMG_annotation/IMG_2737471804/IMG_Data/2737471804/2737471804.cog.tab.txt",
gc_thresh = 0.1, output = FALSE, label = "Bin_2737471804")## Wed Oct 18 15:25:26 2017 --- There are 749 genes with > 0.1 %
## Wed Oct 18 15:25:26 2017 --- This is 100 % of all genes
## Wed Oct 18 15:25:26 2017 --- The 10 genes with the highest GC% are:
## function_id
## 740 Uncharacterized conserved protein, contains von Willebrand factor type A (vWA) domain
## 741 Uncharacterized membrane protein AbrB, regulator of aidB expression
## 742 Hydroxymethylpyrimidine/phosphomethylpyrimidine kinase
## 743 Urease accessory protein UreH
## 744 Rhodanese-related sulfurtransferase
## 745 DNA-binding transcriptional regulator, MarR family
## 746 Geranylgeranyl pyrophosphate synthase
## 747 ABC-type phosphate/phosphonate transport system, permease component
## 748 ABC-type phosphate/phosphonate transport system, ATPase component
## 749 ABC-type phosphate/phosphonate transport system, permease component
## function_name GC
## 740 395 64.2
## 741 352 64.3
## 742 263 64.7
## 743 269 65.1
## 744 110 65.1
## 745 126 65.7
## 746 322 66.3
## 747 283 66.3
## 748 258 67.7
## 749 283 67.9Bin_2737471805_gc_cog <- gc2function(seq_id_gc = "GC_analysis/seqid_GC_133659.assembled.gff.tsv", gene_id_seq_id ="./IMG_annotation/IMG_2737471805/IMG_Data/2737471805/2737471805.gene_oid_2_seq_id.txt",
functions = "./IMG_annotation/IMG_2737471805/IMG_Data/2737471805/2737471805.cog.tab.txt",
gc_thresh = 0.1, output = FALSE, label = "Bin_2737471805")## Wed Oct 18 15:25:26 2017 --- There are 1172 genes with > 0.1 %
## Wed Oct 18 15:25:26 2017 --- This is 100 % of all genes
## Wed Oct 18 15:25:26 2017 --- The 10 genes with the highest GC% are:
## function_id
## 1163 Ribulose-5-phosphate 4-epimerase/Fuculose-1-phosphate aldolase
## 1164 Asp-tRNAAsn/Glu-tRNAGln amidotransferase A subunit or related amidase
## 1165 Biotin-(acetyl-CoA carboxylase) ligase
## 1166 Imidazole glycerol phosphate synthase subunit HisF
## 1167 ABC-type sugar transport system, ATPase component
## 1168 Trk K+ transport system, NAD-binding component
## 1169 Mg-chelatase subunit ChlI
## 1170 NAD(P)-dependent dehydrogenase, short-chain alcohol dehydrogenase family
## 1171 Transcriptional regulator of heat shock response
## 1172 Phosphoheptose isomerase
## function_name GC
## 1163 219 58.2
## 1164 475 58.2
## 1165 238 58.2
## 1166 256 58.3
## 1167 338 58.4
## 1168 225 58.5
## 1169 423 58.8
## 1170 251 58.9
## 1171 346 59.0
## 1172 176 59.2Bin_2737471806_gc_cog <- gc2function(seq_id_gc = "GC_analysis/seqid_GC_133660.assembled.gff.tsv", gene_id_seq_id ="./IMG_annotation/IMG_2737471806/IMG_Data/2737471806/2737471806.gene_oid_2_seq_id.txt",
functions = "./IMG_annotation/IMG_2737471806/IMG_Data/2737471806/2737471806.cog.tab.txt",
gc_thresh = 0.1, output = FALSE, label = "Bin_2737471806")## Wed Oct 18 15:25:26 2017 --- There are 1584 genes with > 0.1 %
## Wed Oct 18 15:25:26 2017 --- This is 100 % of all genes
## Wed Oct 18 15:25:26 2017 --- The 10 genes with the highest GC% are:
## function_id
## 1575 Predicted NAD/FAD-dependent oxidoreductase
## 1576 Gamma-glutamyltranspeptidase
## 1577 Acetyl esterase/lipase
## 1578 CO or xanthine dehydrogenase, FAD-binding subunit
## 1579 3-hydroxyisobutyrate dehydrogenase or related beta-hydroxyacid dehydrogenase
## 1580 Uracil-DNA glycosylase
## 1581 Asp-tRNAAsn/Glu-tRNAGln amidotransferase A subunit or related amidase
## 1582 Predicted secreted protein
## 1583 ABC-type dipeptide/oligopeptide/nickel transport system, ATPase component
## 1584 tRNA A37 threonylcarbamoyladenosine modification protein TsaB
## function_name GC
## 1575 331 67.0
## 1576 539 67.0
## 1577 312 67.2
## 1578 284 67.3
## 1579 286 67.3
## 1580 202 67.4
## 1581 475 67.5
## 1582 148 67.8
## 1583 316 67.8
## 1584 220 69.8merged_gc_cog <- rbind(Bin_2737471681_gc_cog, Bin_2737471682_gc_cog, Bin_2737471683_gc_cog,
Bin_2737471793_gc_cog, Bin_2737471794_gc_cog, Bin_2737471795_gc_cog,
Bin_2737471797_gc_cog, Bin_2737471799_gc_cog, Bin_2737471802_gc_cog,
Bin_2737471804_gc_cog, Bin_2737471805_gc_cog, Bin_2737471806_gc_cog)
merged_gc_cog$genome_id <- as.character(merged_gc_cog$genome_id)4. Analysis of gene length distribution
p_cog_length <- easyGgplot2::ggplot2.histogram(data = merged_gc_cog, xName = 'gene_length',
groupName = 'genome_id', alpha = 0.5,
legendPosition = "top", binwidth = 0.15, addMeanLine=TRUE, meanLineColor="black",
meanLineType="dashed")+ theme_bw()+ ylim(0,15)+
facet_grid(genome_id~.)+
labs(x = "Gene length (bp)", y = "Count")+ theme(legend.position="none", axis.text.x = element_text(angle = 45))+
ggtitle("Limnohabitans MAGs")+ xlim(0,2000)
print(p_cog_length)
5. COG functional categories
Get COG ID to COG functional category mapping file here: ftp://ftp.ncbi.nih.gov/pub/wolf/COGs/COG0303/cogs.csv
The exact statistical analysis to compare genomes based on these profiles should be performed in STAMP.
# Import COG mapping file
cogid_2_cogcat <- read.csv("./Mapping_files/cogid_2_cogcat.csv", sep = ",", header = FALSE, fill = TRUE,col.names = c("COG_ID", "COG_class", "function"))[, 1:2]
cogid_2_cogcat <- cogid_2_cogcat[(cogid_2_cogcat$COG_class)!="", ]
cogid_2_cogcat <- droplevels(cogid_2_cogcat)
# Read COG category file
cog_categories <- read.table("./Mapping_files/cog_categories.tsv", header = TRUE, sep = "\t")
# Merge COG metadata
cog_meta <- dplyr::left_join(cog_categories, cogid_2_cogcat, by = c("COG_class" = "COG_class"))
cog_meta <- droplevels(cog_meta)
# Merge this metadata with the genome data from before
# COGs with multiple classifications are currently still NA - work on this.
merged_gc_cog <- dplyr::left_join(merged_gc_cog, cog_meta, by = c("cog_id" = "COG_ID"))
merged_gc_cog <- merged_gc_cog[!is.na(merged_gc_cog$COG_functional_category),]
# Visualize distribution across major metabolism functional COG groups per genome.
p_cog_func_group <- ggplot(data = merged_gc_cog, aes(x=COG_functional_category, fill = COG_functional_cluster))+
geom_bar(stat="count", width=0.7, color = "black", size = 0.75)+
theme_bw()+
facet_grid(genome_id~.)+
scale_fill_brewer(palette = "Accent")+
labs(x = "Gene length (bp)", y = "Count")+ theme(legend.position="bottom", axis.text.x = element_text(angle = 90, hjust = 1),
legend.text = element_text(size = 7))+
ggtitle("Limnohabitans MAGs")+
guides(fill=guide_legend(nrow=2,byrow=TRUE))
print(p_cog_func_group)
p_cog_func_clust <- ggplot(data = merged_gc_cog, aes(x=COG_functional_cluster, fill = COG_functional_cluster))+
geom_bar(stat="count", width=0.7, color = "black", size = 0.75)+
theme_bw()+
facet_grid(genome_id~.)+
scale_fill_brewer(palette = "Accent")+
labs(x = "Gene length (bp)", y = "Count")+ theme(legend.position="bottom",axis.text.x = element_text(angle = 90, hjust = 1),
legend.text = element_text(size = 7))+
ggtitle("Limnohabitans MAGs")+
guides(fill=guide_legend(nrow=2,byrow=TRUE))
print(p_cog_func_clust)
6. Expression analysis
Remember:
1. Get total nr. of reads through the samtools flagstat command. 2. Get gene length through the DESMAN command: python $DESMAN/scripts/Lengths.py -i CDS.fa > CDS.len
expr_cov <- read.table("./metaT/Coverage_genes.tsv", header = TRUE)
colnames(expr_cov) <- gsub(colnames(expr_cov), pattern = "Map_genes.",
replacement = "")
colnames(expr_cov)[1] <- "gene_oid"
expr_cov$gene_oid <- as.character(expr_cov$gene_oid)
# Merge gene annotations from all genomes in one file
file_list <- list.dirs("./IMG_annotation", recursive = FALSE)
merged_file <- merge_annotations(file_list[1:12], genoid_seqid = TRUE)## --- I will merge the annotation files from the following genomes:
## Genomes
## 1 ./IMG_annotation/IMG_2737471681
## 2 ./IMG_annotation/IMG_2737471682
## 3 ./IMG_annotation/IMG_2737471683
## 4 ./IMG_annotation/IMG_2737471793
## 5 ./IMG_annotation/IMG_2737471794
## 6 ./IMG_annotation/IMG_2737471795
## 7 ./IMG_annotation/IMG_2737471797
## 8 ./IMG_annotation/IMG_2737471799
## 9 ./IMG_annotation/IMG_2737471802
## 10 ./IMG_annotation/IMG_2737471804
## 11 ./IMG_annotation/IMG_2737471805
## 12 ./IMG_annotation/IMG_2737471806
## [1] 3117
## [1] 3117
## [1] 2557
## [1] 5674
## [1] 2456
## [1] 8130
## [1] 2167
## [1] 10297
## [1] 1871
## [1] 12168
## [1] 2061
## [1] 14229
## [1] 1612
## [1] 15841
## [1] 804
## [1] 16645
## [1] 2661
## [1] 19306
## [1] 1157
## [1] 20463
## [1] 1743
## [1] 22206
## [1] 2334
## [1] 24540
## Wed Oct 18 15:26:03 2017 --- Sucessfully merged files# Annotate this expression table with Kegg Orthology and genome ids
expr_cov <- dplyr::left_join(expr_cov, merged_file[, c(1,10)], by = c("gene_oid"))
# Move to long format dataframe for visualization in ggplot2
expr_cov_long <- tidyr::gather(expr_cov, sample, coverage, Fa13.BD.MLB.DN:Su13.BD.MM15.SN,
factor_key = TRUE)
# Calculate average read recruitment to genes (for edgeR/DESeq) and also normalize to transcripts per million (TPM) just in case
# First read in gene lengths and total sample reads
sample_reads_metaT <- read.table("./metaT/sample_reads.tsv", header = TRUE)
gene_lengths_metaT <- read.table("./metaT/genes_concat.len", header = TRUE)
gene_lengths_metaT$gene <- as.character(gene_lengths_metaT$gene)
# Add this information to current long dataframe
expr_cov_long <- dplyr::left_join(expr_cov_long, sample_reads_metaT, by = "sample")
expr_cov_long <- dplyr::left_join(expr_cov_long, gene_lengths_metaT, by = c("gene_oid" = "gene"))
# Now calculate average read recruitment and TPM (relative to the recruitment to these
# genomes in this dataset)
# 150 = read length
expr_cov_long <- dplyr::mutate(expr_cov_long,
mapped_reads = round((coverage * length)/(150),0)
) # Be aware that this average is already rounded here..
expr_cov_long <- dplyr::mutate(expr_cov_long,
reads_per_kb = mapped_reads/length/1000
)
expr_cov_long <- expr_cov_long %>% group_by(sample) %>%
mutate(RPK_scaling = sum(reads_per_kb)/1e6)
expr_cov_long <- expr_cov_long %>%
mutate(TPM = reads_per_kb/RPK_scaling)
# Now add the metadata to this long dataframe
expr_cov_long <- left_join(expr_cov_long, meta[, 1:11], by = c("sample" = "Sample"))
expr_cov_long$Genome_id <- as.factor(expr_cov_long$Genome_id)
# Remove duplicate rows
expr_cov_long <- expr_cov_long %>% distinct()# Format data for DESeq2
## Put count matrices in list including a count matrix for each bin
expr_cov_bins <- list()
for(i in 1:nlevels(expr_cov_long$Genome_id)){
expr_cov_bins[[i]] <- expr_cov_long %>%
filter(Genome_id == levels(expr_cov_long$Genome_id)[i]) %>%
select(gene_oid, sample, mapped_reads) %>%
spread(sample, mapped_reads)
r.bin <- expr_cov_bins[[i]]$gene_oid
expr_cov_bins[[i]] <- as.matrix(expr_cov_bins[[i]][, -1])
rownames(expr_cov_bins[[i]]) <- r.bin
}
# Metadata file
meta_metaT <- distinct(meta[, 2:nrow(meta)])
rownames(meta_metaT) <- gsub(meta_metaT$Sample_ID, pattern="_", replacement = ".")
meta_metaT <- as.matrix(meta_metaT)
# Check order of colnames in count and rownames in metadata matrix
# and make sure these are in the same order!
meta_metaT <- meta_metaT[match(colnames(expr_cov_bins[[1]]), rownames(meta_metaT)), ]
all(rownames(meta_metaT) %in% colnames(expr_cov_bins[[1]]))## [1] TRUE# Perform DESeq2 for differential abundance testing for each genome separately
## Season effect
General_deseq_results_season <- list()
for(i in 1:nlevels(expr_cov_long$Genome_id)){
cat(" --- Running DESeq2 on Genome_id:",levels(expr_cov_long$Genome_id)[i],sep = " ")
dds <- DESeqDataSetFromMatrix(countData = expr_cov_bins[[i]],
colData = meta_metaT,
design= ~ Site + Season) # Test for season but controlling for site
dds <- DESeq(dds)
General_deseq_results_season[[i]] <- results(dds)[order(results(dds)$padj), ] # specify contrasts here if need to
summary(results(dds, alpha=0.05))
}## --- Running DESeq2 on Genome_id: 2737471681## converting counts to integer mode## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testing##
## out of 3117 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 30, 0.96%
## LFC < 0 (down) : 71, 2.3%
## outliers [1] : 22, 0.71%
## low counts [2] : 61, 2%
## (mean count < 1)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results
##
## --- Running DESeq2 on Genome_id: 2737471682## converting counts to integer mode## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testing##
## out of 2555 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 4, 0.16%
## LFC < 0 (down) : 21, 0.82%
## outliers [1] : 14, 0.55%
## low counts [2] : 0, 0%
## (mean count < 0)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results
##
## --- Running DESeq2 on Genome_id: 2737471683## converting counts to integer mode## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testing##
## out of 2455 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 2, 0.081%
## LFC < 0 (down) : 1, 0.041%
## outliers [1] : 16, 0.65%
## low counts [2] : 0, 0%
## (mean count < 0)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results
##
## --- Running DESeq2 on Genome_id: 2737471793## converting counts to integer mode## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testing##
## out of 2167 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 1, 0.046%
## LFC < 0 (down) : 2, 0.092%
## outliers [1] : 14, 0.65%
## low counts [2] : 0, 0%
## (mean count < 0)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results
##
## --- Running DESeq2 on Genome_id: 2737471794## converting counts to integer mode## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testing##
## out of 1871 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 0, 0%
## LFC < 0 (down) : 1, 0.053%
## outliers [1] : 9, 0.48%
## low counts [2] : 0, 0%
## (mean count < 0)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results
##
## --- Running DESeq2 on Genome_id: 2737471795## converting counts to integer mode## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testing##
## out of 2061 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 3, 0.15%
## LFC < 0 (down) : 4, 0.19%
## outliers [1] : 15, 0.73%
## low counts [2] : 0, 0%
## (mean count < 0)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results
##
## --- Running DESeq2 on Genome_id: 2737471797## converting counts to integer mode## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testing##
## out of 1611 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 11, 0.68%
## LFC < 0 (down) : 30, 1.9%
## outliers [1] : 16, 0.99%
## low counts [2] : 437, 27%
## (mean count < 3)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results
##
## --- Running DESeq2 on Genome_id: 2737471799## converting counts to integer mode## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testing##
## out of 804 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 1, 0.12%
## LFC < 0 (down) : 6, 0.75%
## outliers [1] : 9, 1.1%
## low counts [2] : 0, 0%
## (mean count < 0)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results
##
## --- Running DESeq2 on Genome_id: 2737471802## converting counts to integer mode## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testing##
## out of 2657 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 11, 0.41%
## LFC < 0 (down) : 13, 0.49%
## outliers [1] : 24, 0.9%
## low counts [2] : 1330, 50%
## (mean count < 8)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results
##
## --- Running DESeq2 on Genome_id: 2737471804## converting counts to integer mode## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testing##
## out of 1157 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 7, 0.61%
## LFC < 0 (down) : 6, 0.52%
## outliers [1] : 6, 0.52%
## low counts [2] : 202, 17%
## (mean count < 3)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results
##
## --- Running DESeq2 on Genome_id: 2737471805## converting counts to integer mode## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testing##
## out of 1743 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 1, 0.057%
## LFC < 0 (down) : 0, 0%
## outliers [1] : 8, 0.46%
## low counts [2] : 0, 0%
## (mean count < 0)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results
##
## --- Running DESeq2 on Genome_id: 2737471806## converting counts to integer mode## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testing##
## out of 2334 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 2, 0.086%
## LFC < 0 (down) : 4, 0.17%
## outliers [1] : 18, 0.77%
## low counts [2] : 0, 0%
## (mean count < 0)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results## Site effect
General_deseq_results_site <- list()
for(i in 1:nlevels(expr_cov_long$Genome_id)){
cat(" --- Running DESeq2 on Genome_id:",levels(expr_cov_long$Genome_id)[i],sep = " ")
dds <- DESeqDataSetFromMatrix(countData = expr_cov_bins[[i]],
colData = meta_metaT,
design= ~ Season + Site) # Test for season but controlling for site
dds <- DESeq(dds)
General_deseq_results_site[[i]] <- results(dds)[order(results(dds)$padj), ] # specify contrasts here if need to
summary(results(dds, alpha=0.05))
}## --- Running DESeq2 on Genome_id: 2737471681## converting counts to integer mode## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testing##
## out of 3117 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 356, 11%
## LFC < 0 (down) : 140, 4.5%
## outliers [1] : 22, 0.71%
## low counts [2] : 0, 0%
## (mean count < 0)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results
##
## --- Running DESeq2 on Genome_id: 2737471682## converting counts to integer mode## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testing##
## out of 2555 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 335, 13%
## LFC < 0 (down) : 218, 8.5%
## outliers [1] : 14, 0.55%
## low counts [2] : 100, 3.9%
## (mean count < 1)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results
##
## --- Running DESeq2 on Genome_id: 2737471683## converting counts to integer mode## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testing##
## out of 2455 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 98, 4%
## LFC < 0 (down) : 195, 7.9%
## outliers [1] : 16, 0.65%
## low counts [2] : 284, 12%
## (mean count < 3)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results
##
## --- Running DESeq2 on Genome_id: 2737471793## converting counts to integer mode## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testing##
## out of 2167 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 76, 3.5%
## LFC < 0 (down) : 137, 6.3%
## outliers [1] : 14, 0.65%
## low counts [2] : 294, 14%
## (mean count < 5)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results
##
## --- Running DESeq2 on Genome_id: 2737471794## converting counts to integer mode## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testing##
## out of 1871 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 88, 4.7%
## LFC < 0 (down) : 202, 11%
## outliers [1] : 9, 0.48%
## low counts [2] : 72, 3.8%
## (mean count < 2)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results
##
## --- Running DESeq2 on Genome_id: 2737471795## converting counts to integer mode## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testing##
## out of 2061 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 129, 6.3%
## LFC < 0 (down) : 260, 13%
## outliers [1] : 15, 0.73%
## low counts [2] : 118, 5.7%
## (mean count < 2)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results
##
## --- Running DESeq2 on Genome_id: 2737471797## converting counts to integer mode## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testing##
## out of 1611 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 256, 16%
## LFC < 0 (down) : 101, 6.3%
## outliers [1] : 16, 0.99%
## low counts [2] : 94, 5.8%
## (mean count < 1)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results
##
## --- Running DESeq2 on Genome_id: 2737471799## converting counts to integer mode## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testing##
## out of 804 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 39, 4.9%
## LFC < 0 (down) : 63, 7.8%
## outliers [1] : 9, 1.1%
## low counts [2] : 78, 9.7%
## (mean count < 1)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results
##
## --- Running DESeq2 on Genome_id: 2737471802## converting counts to integer mode## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testing##
## out of 2657 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 135, 5.1%
## LFC < 0 (down) : 202, 7.6%
## outliers [1] : 24, 0.9%
## low counts [2] : 664, 25%
## (mean count < 4)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results
##
## --- Running DESeq2 on Genome_id: 2737471804## converting counts to integer mode## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testing##
## out of 1157 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 244, 21%
## LFC < 0 (down) : 198, 17%
## outliers [1] : 6, 0.52%
## low counts [2] : 0, 0%
## (mean count < 0)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results
##
## --- Running DESeq2 on Genome_id: 2737471805## converting counts to integer mode## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testing##
## out of 1743 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 237, 14%
## LFC < 0 (down) : 373, 21%
## outliers [1] : 8, 0.46%
## low counts [2] : 0, 0%
## (mean count < 0)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results
##
## --- Running DESeq2 on Genome_id: 2737471806## converting counts to integer mode## estimating size factors## estimating dispersions## gene-wise dispersion estimates## mean-dispersion relationship## final dispersion estimates## fitting model and testing##
## out of 2334 with nonzero total read count
## adjusted p-value < 0.05
## LFC > 0 (up) : 319, 14%
## LFC < 0 (down) : 202, 8.7%
## outliers [1] : 18, 0.77%
## low counts [2] : 0, 0%
## (mean count < 0)
## [1] see 'cooksCutoff' argument of ?results
## [2] see 'independentFiltering' argument of ?results##
##
sessionInfo()## R version 3.4.1 (2017-06-30)
## Platform: x86_64-w64-mingw32/x64 (64-bit)
## Running under: Windows 8.1 x64 (build 9600)
##
## Matrix products: default
##
## locale:
## [1] LC_COLLATE=English_United States.1252
## [2] LC_CTYPE=English_United States.1252
## [3] LC_MONETARY=English_United States.1252
## [4] LC_NUMERIC=C
## [5] LC_TIME=English_United States.1252
##
## attached base packages:
## [1] parallel stats4 grid stats graphics grDevices utils
## [8] datasets methods base
##
## other attached packages:
## [1] bindrcpp_0.2 SpiecEasi_0.1.2
## [3] DESeq2_1.12.4 SummarizedExperiment_1.2.3
## [5] Biobase_2.32.0 GenomicRanges_1.24.3
## [7] GenomeInfoDb_1.8.7 IRanges_2.6.1
## [9] S4Vectors_0.10.3 BiocGenerics_0.18.0
## [11] igraph_1.1.2 phyloseq_1.16.2
## [13] edgeR_3.14.0 limma_3.28.21
## [15] easyGgplot2_1.0.0.9000 gridExtra_2.3
## [17] ggplot2_2.2.1 dplyr_0.7.4
## [19] tidyr_0.6.0 knitr_1.17
##
## loaded via a namespace (and not attached):
## [1] nlme_3.1-131 bit64_0.9-7 RColorBrewer_1.1-2
## [4] rprojroot_1.2 tools_3.4.1 backports_1.1.1
## [7] R6_2.2.2 vegan_2.4-4 rpart_4.1-11
## [10] Hmisc_4.0-3 DBI_0.7 lazyeval_0.2.0
## [13] mgcv_1.8-22 colorspace_1.3-2 permute_0.9-4
## [16] ade4_1.7-8 nnet_7.3-12 bit_1.1-12
## [19] compiler_3.4.1 htmlTable_1.9 labeling_0.3
## [22] scales_0.5.0 checkmate_1.8.4 genefilter_1.54.2
## [25] stringr_1.2.0 digest_0.6.12 foreign_0.8-69
## [28] rmarkdown_1.6 XVector_0.12.1 base64enc_0.1-3
## [31] pkgconfig_2.0.1 htmltools_0.3.6 htmlwidgets_0.9
## [34] rlang_0.1.2 huge_1.2.7 VGAM_1.0-4
## [37] RSQLite_2.0 bindr_0.1 jsonlite_1.5
## [40] BiocParallel_1.6.6 acepack_1.4.1 magrittr_1.5
## [43] Formula_1.2-2 biomformat_1.0.2 Matrix_1.2-11
## [46] Rcpp_0.12.13 munsell_0.4.3 ape_4.1
## [49] stringi_1.1.5 yaml_2.1.14 MASS_7.3-47
## [52] zlibbioc_1.18.0 rhdf5_2.16.0 plyr_1.8.4
## [55] blob_1.1.0 lattice_0.20-35 Biostrings_2.40.2
## [58] splines_3.4.1 multtest_2.28.0 annotate_1.50.0
## [61] locfit_1.5-9.1 boot_1.3-20 geneplotter_1.50.0
## [64] reshape2_1.4.2 codetools_0.2-15 XML_3.98-1.9
## [67] glue_1.1.1 evaluate_0.10.1 latticeExtra_0.6-28
## [70] data.table_1.10.4 foreach_1.4.3 gtable_0.2.0
## [73] assertthat_0.2.0 xtable_1.8-2 survival_2.41-3
## [76] tibble_1.3.4 iterators_1.0.8 memoise_1.1.0
## [79] AnnotationDbi_1.34.4 cluster_2.0.67. Sequence discrete populations
In order to check unspecific mapping sample reads were mapped consecutively to every bin using BBmap.sh. This approach allows us to check the mapping specificity by evaluating the distribution of read identity to the putative genome bin.
map_disc <- read.table("./SEQs_discrete/final.idhist", header = FALSE,
row.names = NULL)
colnames(map_disc) <- c("bin","sample", "identity", "reads_mapped", "bases_mapped")
# Add season metadata
map_disc$season <- "Summer"
map_disc$season[grep("Fa", map_disc$sample)] <- "Fall"
map_disc$season[grep("Su", map_disc$sample)] <- "Summer"
map_disc$season[grep("Sp", map_disc$sample)] <- "Spring"
map_disc$sample <- gsub(".C","", map_disc$sample, fixed = TRUE)
total_reads2 <- total_reads
total_reads2$sample <- gsub("_", ".", fixed = TRUE,total_reads$sample)
map_disc <- dplyr::left_join(map_disc, total_reads2, by = "sample")
# Throw away all %identity below 60%
# map_disc <- map_disc %>% filter(identity > 60)
# Normalize reads_mapped to library sizes
map_disc <- map_disc %>% group_by(sample) %>%
mutate(rel_reads_mapped = 100*reads_mapped/Total_reads)
# Plot
for(bin2plot in unique(map_disc$bin)){
p_sdisc <- map_disc %>% filter(bin == bin2plot) %>%
ggplot(aes(x = identity, y = rel_reads_mapped, color = season))+
theme_bw()+
scale_color_brewer(palette = "Accent")+
facet_wrap(~sample, nrow = 4)+
geom_line(size = 1.5)+
guides(color = FALSE)+
ggtitle(bin2plot)+
theme(axis.text=element_text(size=14), axis.title=element_text(size=20),
title=element_text(size=20), legend.text=element_text(size=14),
legend.background = element_rect(fill="transparent"),
axis.text.x = element_text(angle = 45, hjust = 1),
strip.text.y=element_text(size=14))+
ylab("Proportion of reads mapped (%)")+
xlab("Nucleotide identity (%)")
# ylim(0,1.5)
print(p_sdisc)
}
# Plot % reads over threshold of 0.95
id_thresh <- 95
map_disc_cum <- map_disc %>% filter(identity > id_thresh) %>% group_by(sample) %>%
mutate(cum_rel_reads_mapped = cumsum(rel_reads_mapped))%>%
filter(identity == 100)
p_sdisc_cum <- ggplot(map_disc_cum, aes(x = sample, y = cum_rel_reads_mapped,
fill = bin))+
theme_bw()+
scale_fill_brewer(palette = "Paired")+
geom_point(size = 4, shape = 21, color = "black")+
theme(axis.text=element_text(size=14), axis.title=element_text(size=20),
title=element_text(size=20), legend.text=element_text(size=14),
legend.background = element_rect(fill="transparent"),
axis.text.x = element_text(angle = 45, hjust = 1),
strip.text.y=element_text(size=14), legend.position = "bottom")+
ylab(paste0("Proportion of reads mapped > ", id_thresh, "% NI"))+
xlab("Sample")+
guides(fill=guide_legend(nrow = 11))
print(p_sdisc_cum)